Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing is widely used to understand the biological processes at a single cell level. The advent of multiplexing strategies has further expanded its amplifications by allowing the profiling of multiple samples in a single experiment, dramatically reducing the cost and avoiding the batch effects. Demonstrating the procedure will be Wei Feng, a postdoc, and Andrew Przysinda, a technician from my lab.

After counting, split the cells isolated from embryonic day 18.5 heart chambers into five times 10 to the fifth aliquots and wash the cells two times with fresh PBS per wash. Resuspend the pellets in 180 microliters of PBS per sample and add 20 microliters of anchor barcode stock solution to each tube with gentle mixing. After a five-minute incubation on ice, add 20 microliters of co-anchor barcode stock solution to each tube with gentle mixing for an additional five-minute incubation on ice before washing the cells three times with one milliliter of cold PBS supplemented with 1%bovine serum albumin per wash, then filter the samples into new tubes through one 40-micrometer cell strainer per tube.

After counting, assemble chip B into a chip holder, then add 75 microliters of a 50%glycerol solution to the unused wells in row one, 40 microliters to the unused wells in row two, and 280 microliters to the unused wells in row three. Note that a training chip is used here to demonstrate the procedure. Add the appropriate volume of cell suspension and nuclease-free water to the master mix with gentle pipetting.

Add 75 microliters of the resulting cell solution to the bottom center of the sample well in row one without bubbles. Vortex the gel beads for 30 seconds before slowly adding 40 microliters of beads to the bottom center of the gel bead well in row two without bubbles. Add 280 microliters of partitioning oil down the sidewall of the partitioning oil well in row three.

Attach the gasket to the chip without pressing down on the gasket and keeping the gasket horizontal to avoid wetting the gasket. Load the assembled chip with the gasket in the chromium controller and run the chromium single cell B program. Note that the screen shows chromium training for the training chip but for the real experiments the screen will display chromium single cell B.When the program has completed, immediately remove the chip and discard the gasket.

Fold the lid back to expose the wells at a 45-degree angle and check the liquid levels to make sure no clogs are present. Slowly aspirate 100 microliters of gel beads in emulsion from the lowest points of the recovery well and check the uniformity of the beads. Dispense the emulsified beads down the wall of a new PCR tube on ice and place the tube in a thermal cycler for reverse transcription.

To prepare the cDNA for amplification, add 125 microliters of recovery agent to the sample at room temperature. No opaque liquid should be observed and avoid pipetting or vortexing. Wait 60 seconds before slowly removing the recovery agent from the bottom of the tube and add 200 microliters of vortex bead cleanup mixture to the sample.

Pipette the mixture 10 times before incubating for 10 minutes at room temperature. Next, place the samples onto a magnet in the high position. When the solution clears, remove the supernatant and add 200 microliters of 80%ethanol to the pellet.

After 30 seconds, remove the ethanol and repeat the wash two more times. After the last wash, briefly centrifuge the sample and place the tube on the magnet in the low position to allow the sample to air dry for less than two minutes. When the sample has dried, remove the tube from the magnet and add 35.5 microliters of freshly prepared elution solution.

After a two-minute incubation at room temperature, place the sample on the magnet in the high position until the solution clears before transferring 35 microliters of the sample to a new tube strip. For cDNA amplification using the lipid-based barcoding strategy, add amplification reaction mixture to the 35-microliter cDNA samples with thorough mixing before briefly centrifuging. At the end of the spin, incubate the sample in the thermal cycler following the appropriate cDNA amplification procedure.

Add 120 microliters of select reagent and 100 microliters of ultrapure water to 100 microliters of sample and pipette the resulting 0.6x select reagent 15 times. After a five-minute incubation at room temperature, place the sample on the magnet until the solution becomes clear. Transfer the supernatant to a 1.5-milliliter low bind tube for multiplexing barcoded cDNA library construction.

Remember to save both the supernatants and the base as the supernatant's contents assemble barcoded cDNA and the base contents endogenous cDNA. Clean the endogenous cDNA with 80%ethanol and elute the DNA with 40 microliters of elution buffer, then run a quality control check of the endogenous cDNA before preparing an endogenous transcript library. The cDNA concentration should be quantified and qualified before library construction.

The constructed libraries, including the endogenous cDNA and barcode libraries, should also be quantified and qualified before sequencing. After sequencing, the barcode expression can be analyzed in each single cell. For example, in this analysis eight groups of single cells that uniquely expressed one type of barcode representing cells from eight different samples were observed.

In addition, some cells did not express any barcode and therefore were defined as negative cells, while other cells expressed two different barcodes representing doublets. Using the singlet cells, the cellular heterogeneity and molecular regulations could be assessed by cell type annotation, novel and rare cell type identification, anatomical zone comparative analysis, and gene ontology pathway analysis, such as cell cycle phase separations. It is helpful to image the gel beads in emulsion if you can, as an image will tell you whether or not the procedure has been successful up to this point.

The sample multiplexing adds great flexibility in designing your experiments. After watching this demonstration, I hope you will be more confident about starting your own experiments.